The binding of methotrexate, one of the most widely used and effective anti-cancer chemotherapeutic agents, to its target site liver dihydrofolate reductase was studied by measuring the hearts of binding. The enthalpy of methotrexate-enzyme interaction demonstrates that a proton transfer occurs upon binding whereas this is not the case with the binding of the natural vitamin cofactors, folic acid or dihydrofolic acid. This finding confirms the speculations derived from quantum mechanical calculations and the conclusions drawn from the difference spectra characteristic of methotrexate interaction with dihydrofolate reductase. In addition, the thermodynamic parameters characterizing the tight binding of methotrexate, folates and pyridine nucleotides to chicken liver dihydrofolate reductase have been determined. The binding of cosubstrates NADPH and NADP ions is characterized by small negative enthalpies and large positive entropies while the binding of the folates as well as methotrexate is accompanied by large negative enthalpies and small negative entropies. These results suggest that the binding of the pyridine nucleotide cofactors is quite different from that of the binding of the folates in terms of the interactions at the active site of the protein.